Cytochromes P450 (CYP) are the principal enzymes for the oxidative metabolism of many drugs, procarcinogens, promutagens, and environmental pollutants. Cytochrome P450 is a heme-containing, membrane-bound, multienzyme system that is present in many tissues in vivo but is present at the highest level in liver. In human liver, it is estimated that there are 15-20 different xenobiotic-metabolizing cytochrome P450 forms. A standard nomenclature based on relatedness of amino acid sequences has been developed. Certain P450 forms (such as CYP2D6 and CYP2C19) are known to be polymorphic in humans and some (such as CYP1A2 and CYP3A4) are regulated in response to environmental chemicals. Competition for metabolism by a particular cytochrome P450 form is a principal mechanism of some clinically significant drug--drug interactions.
Identification of the enzymes responsible for metabolism is becoming an important aspect of drug development. Such identifications consider both the metabolism of the new drug as well as inhibition by the new drug. The identification of enzymes involved in metabolism of the new drug allows prediction, based on knowledge of the ability of coadministered drugs to inhibit the same enzymes, of which coadministered drugs may inhibit the metabolism of the new drug. This information can also be used to predict individual variability based on known metabolic polymorphisms. The identification of the enzymes most sensitive to inhibition by the new drug allows prediction, based on knowledge of which coadministered drugs are metabolized by the same enzyme, of which coadministered drug's metabolism may be inhibited by the new drug. Obtaining information for a series of drug candidates early in the drug discovery process can assist in the choice of the best drug candidate for further development.
CYP2D6 is the only member of the CYP2D subfamily that is expressed in humans. CYP2D6 is responsible for the metabolism of many important drugs, for example: cough suppressants, antiarrhythmics, and psychotropic drugs. CYP2D6 is also polymorphic. About 5-10% of Caucasians and 1-3% of Asians and Africans are deficient in this enzyme. About 5% of Caucasians are ultrarapid metabolizers, have very high levels of CYP2D6 (S. Rendic and F. J. Di Carlo, Drug Metab. Rev. 29, 413-580 (1997)). The importance of this P450 in drug metabolism, as well as its variability in activity due to polymorphisms, makes the screening for metabolism and inhibition of this enzyme important in drug development.
Assays for CYP2D have focused on the metabolism of drug molecules or drug candidates. Several drugs are reported to be metabolized by CYP2D6 via O-dealkylation. For example, dextromethorphan is O-demethylated by CYP2D6 (Kupfer, A., Schmid, B., Preisig, R., and G. Pfaff (1984) Lancet i, p517.). Substrates such as dextromethorphan or bufuralol are effective in assessing CYP2D activity and inhibition, but are not amenable to high throughput screening assay technology (both require time consuming separation of CYP2D reaction products using HPLC). Also, neither of these substrates has the necessary fluorescent properties that make the substrate useful for in situ fluorescent plate analysis.
We have previously reported the use of the commercially available compound 3-cyano-7-ethoxycoumarin (CEC) as a fluorescent substrate for assessing CYP2D6 activity in a high throughput mode (See Crespi et al. Anal Biochem. 248, 188-190, (1997). However, the low enzymatic turnover and poor specificity of this substrate make it of limited utility.